Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated ...N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 unique peptides, we characterized N-terminal methionine excision, co- and posttranslational Nα acetylation, protein maturation, and proteolytic processing of proteins in human platelets. We also identified for the first time 10 proteins previously classified by the Human Proteome Organization as “missing” in the human proteome. Most N termini (77%) were internal neo-N termini (105 were novel potential alternative translation start sites, and 2180 represented stable proteolytic products), thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metalloproteinase and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All data sets and metadata are available through ProteomeXchange with the data set identifier PXD000906.
•TAILS proteomics identified 2938 human platelet proteins, pervasive proteolytic processing, and precise proteolytic cleavage sites in stored platelets.•During storage, metalloproteinases were predominantly involved in proteolytic processing, while other proteinases were mainly involved in degradation.
Background
Equitable allocation of scarce blood products needed for a randomized controlled trial (RCT) is a complex decision‐making process within the blood supply chain. Strategies to improve ...resource allocation in this setting are lacking.
Methods
We designed a custom‐made, computerized system to manage the inventory and allocation of COVID‐19 convalescent plasma (CCP) in a multi‐site RCT, CONCOR‐1. A hub‐and‐spoke distribution model enabled real‐time inventory monitoring and assignment for randomization. A live CCP inventory system using REDCap was programmed for spoke sites to reserve, assign, and order CCP from hospital hubs. A data‐driven mixed‐integer programming model with supply and demand forecasting was developed to guide the equitable allocation of CCP at hubs across Canada (excluding Québec).
Results
18/38 hospital study sites were hubs with a median of 2 spoke sites per hub. A total of 394.5 500‐ml doses of CCP were distributed; 349.5 (88.6%) doses were transfused; 9.5 (2.4%) were wasted due to mechanical damage sustained to the blood bags; 35.5 (9.0%) were unused at the end of the trial. Due to supply shortages, 53/394.5 (13.4%) doses were imported from Héma‐Québec to Canadian Blood Services (CBS), and 125 (31.7%) were transferred between CBS regional distribution centers to meet demand. 137/349.5 (39.2%) and 212.5 (60.8%) doses were transfused at hubs and spoke sites, respectively. The mean percentages of total unmet demand were similar across the hubs, indicating equitable allocation, using our model.
Conclusion
Computerized tools can provide efficient and immediate solutions for equitable allocation decisions of scarce blood products in RCTs.
BACKGROUND
Biochemical analyses of mechanisms triggered in platelets (PLTs) upon pathogen inactivation (PI) are crucial to further understand the impact of PI on PLT functionality and, subsequently, ...quality.
STUDY DESIGN AND METHODS
PLT concentrates (PCs) were split into four small illumination bags: 1) untreated control, 2) treated with riboflavin and ultraviolet light (RF/UV), and spiked with 3) solvent control dimethyl sulfoxide and 4) p38 mitogen‐activated protein kinase (MAPK) inhibitor SB203580 before RF/UV treatment. Flow cytometry was used to monitor PLT mitochondrial potential (ΔΨm); generation of intracellular reactive oxygen species (ROS); and release of microvesicles (MVs), mitochondria (MT), and MVs containing MT (MVs/MT). Quantitative polymerase chain reaction (qPCR) was used to quantify extracellular mitochondrial DNA (mtDNA). Translocation of selected mitochondrial proteins was analyzed in subcellular fractions by immunoblot.
RESULTS
RF/UV treatment triggered an increased mitochondrial translocation of both Bax and Bid (p < 0.05, Day 7) and cytochrome c release (p < 0.01, Day 7), loss of ΔΨm (p < 0.05, Day 5 and Day 7), and ROS generation (p < 0.01, Day 5 and Day 7) in PCs compared to the untreated control during storage. These PI‐triggered changes were inhibited by SB203580 (p < 0.05). The release of MVs, MT, and MVs/MT was increased upon the RF/UV treatment during storage (p < 0.05) and, with the exception of MT, the release was decreased by the inhibitor (p < 0.05). qPCR analysis showed that RF/UV does not trigger mtDNA release during storage.
CONCLUSION
These findings further our understanding of mechanisms in PLTs initiated by the RF/UV treatment, demonstrating that this treatment induces p38 MAPK‐dependent mitochondrial signaling and MV release in apheresis PCs.
Background
Transfusion medicine standards in Canada state that adult recipients can be transfused with cryoprecipitate of any ABO group, however, not all hospitals follow this guideline. There is a ...paucity of data on cryoprecipitate anti‐A/B levels to reinforce standards.
Study Design and Methods
Manual tube antibody titration was performed on 7 units of group O plasma and the corresponding cryosupernatant plasma and cryoprecipitate. IgG/IgM levels were determined by nephelometry. Additionally, 10 cryoprecipitate each from groups A, B, and O were similarly assessed. From the antibody titer distribution among these samples, the probability of making a pool of cryoprecipitate with a titer ≥1:100 was calculated using bootstrap analysis.
Results
Anti‐A/B titers in cryoprecipitate were equivalent to those in corresponding plasma; partitioning of anti‐A/B activity into cryoprecipitate was not observed. Average IgM concentration was higher in cryoprecipitate than in plasma (P < .01). However, no correlation between IgM levels and anti‐A/B titers was established. Among 30 cryoprecipitates from routine blood bank inventory, the median antibody titer and mode were 1:32 and 1:16, respectively. Of the samples tested, 4 of 30 and 9 of 30 had titers above 1:100 and 1:50, respectively. The probability of transfusing an adult dose of cryoprecipitate (pool of 10 cryoprecipitate) with a titer higher than 1:100 was calculated to be less than 1 in 3 million.
Conclusions
This study provides strong evidence to support current Canadian transfusion medicine standards on the safety of transfusion of cryoprecipitate without the need for blood group matching in adult recipients.
See editorial on page 1–4, in this issue
50 years of Vox Sanguinis International Forums Lozano, Miquel; Dunbar, Nancy M.; Devine, Dana V.
Vox sanguinis,
August 2020, 2020-Aug, 2020-08-00, 20200801, Volume:
115, Issue:
6
Journal Article
Keywords: blood center operations; FFP transfusion; transfusion practices (adult) Byline: Claudia S. Cohn, Lise Estcourt, Brenda J. Grossman, Monica B. Pagano, Elizabeth S. Allen, Evan M. Bloch, ...Arturo Casadevall, Dana V. Devine, Nancy M. Dunbar, Farid Foroutan, Thomas J. Gniadek, Ruchika Goel, Jed Gorlin, Michael J. Joyner, Ryan A. Metcalf, Jay S. Raval, Todd W. Rice, Beth H. Shaz, Ralph R. Vassallo, Jeffrey L. Winters, Gregory Beaudoin, Aaron A. R. Tobian
Adsorbing toxins from the blood to augment membrane-based hemodialysis is an active area of research. Films composed of β-cyclodextrin-co-(methacryloyloxy)ethyl phosphorylcholine (p(PMβCD-co-MPC)) ...with various monomer ratios were formed on magnetic nanoparticles and characterized. Surface chemistry effects on protein denaturation were evaluated and indicated that unmodified magnetic nanoparticles greatly perturbed the structure of proteins compared to coated particles. Plasma clotting assays were conducted to investigate the stability of plasma in the presence of particles, where a 2:2 monomer ratio yielded the best results for a given total surface area of particles. Total protein adsorption results revealed that modified surfaces exhibited reduced protein adsorption compared to bare particles, and pure MPC showed the lowest adsorption. Immunoblot results showed that fibrinogen, α1-antitrypsin, vitronectin, prekallikrein, antithrombin, albumin, and C3 correlated with film composition. Hemocompatibility testing with whole blood illustrated that the 1:3 ratio of CD to MPC had a negative impact on platelets, as evidenced by the increased activation, reduced response to an agonist, and reduced platelet count. Other formulations had statistically significant effects on platelet activation, but no formulation yielded apparent adverse effects on hemostasis. For the first time, p(PMβCD-co-MPC)-coated MNP were synthesized and their general hemocompatibility assessed.
BACKGROUND
In neonate transfusion, the use of a dedicated red blood cell (RBC) unit decreases donor exposure. A separate safety measure involves gamma irradiation of the RBCs to abrogate the ...possibility of transfusion‐associated graft‐versus‐host disease. However, in combination, storage of gamma‐irradiated RBCs leads to accumulation of potentially harmful substances in the supernatant.
STUDY DESIGN AND METHODS
For this study, RBCs were pooled and split into three study arms. Centrifugation or gravity was used to pack RBCs of matched units thereby reducing the amount of supernatant that would be present in neonate transfusion aliquots; these were compared to matched control units. Supernatant measurements of potassium, hemoglobin (Hb), RBC microvesicle (RMV) content, and mannitol were made in aliquots prepared weekly up to 21 days after gamma irradiation. RBC morphology and osmotic fragility were also assessed to determine if supernatant reduction methods affected the storage lesion.
RESULTS
Potassium and mannitol were significantly decreased in transfusion aliquots prepared with either of the supernatant reduction methods. On Day 21, potassium levels from supernatant‐reduced aliquots were below those of Day 7 control aliquots. A decrease in free Hb was only detected on Day 21 in centrifuged aliquots. RMVs were significantly reduced in centrifuged aliquots and significantly increased in gravity‐settled aliquots. The only measurable effect on storage lesion was a small increase in osmotic fragility of the RBCs subjected to supernatant reduction.
CONCLUSION
Supernatant reduction by centrifugation effectively reduces potassium, mannitol, and RMVs in aliquots from gamma‐irradiated RBCs stored up to 21 days.
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